ABSTRACT Rickettsia are obligate intracellular bacteria of the a-proteobacteria family impacting human development as causative agents of Typhus and Spotted Fever. Hallmark of Rickettsia is their replication in human vascular endothelial cells, release into the bloodstream and transmission via hematophagic arthropods, with Rickettsia replicating in the gastrointestinal tract or salivary glands of various vectors (lice, ticks, mites or fleas). Owing to technical difficulties of isolating and propagating Rickettsia, Typhus and Spotted Fever have long been diagnosed via serological agglutination for antibodies cross-reactive with Proteus vulgaris (Weil-Felix reaction). For epidemic Typhus, positive Weil-Felix serology is associated with survival and with the development of immunity that, if waning, may precipitate recrudescent typhus with recall of Weil-Felix serology. Early work developed vaccines successful at preventing epidemic Typhus, however the mechanisms for protective immunity are still unknown. To explore the genetic bases of rickettsial pathogenesis, several laboratories developed technologies for generating mutations in Rickettsia. This work identified mutants with defects in actin-based motility and cell-to-cell spread. Nevertheless, genes underpinning rickettsial entry into vascular endothelial cells, intracellular replication, release into the bloodstream or replication in various cell types of their insect vectors remain largely unknown. Here we describe an in vitro transposition reaction using purified Tn5-transposase complexed with mini-transposon, which provides for chloramphenicol-selection of Rickettsia conorii variants with chromosomal insertions. DNA sequencing of insertion sites demonstrates the random nature of insertional mutagenesis with the mini-transposon, while chloramphenicol-selection allows for facile isolation of variants that can be analyzed for in vitro replication as well as defects in the pathogenesis of Boutonneuse Fever. One of the isolated R. conorii variants, with insertion in the polysaccharide synthesis operon (pso), exhibits defects in attachment and invasion of tissue culture cells and in Spotted Fever pathogenesis in mice. The disrupted gene encodes an enzyme predicted to synthesize QuiNAc, a structural determinant in the lipopolysaccharide of P. vulgaris OX19. As the pso locus encompasses genes that are conserved or variable among different rickettsial species, these data suggest that lipopolysaccharide of R. conorii contributes to the Rickettsia replicative life-style, elicits immune responses that define serological cross- reactivity with P. vulgaris (Weil-Felix serology) and may represent a determinant of antibody-mediated immunity. To further the genetic analysis of Rickettsia, we propose to use insertional mutagenesis to generate a library of R. conorii transposon mutants. These mutants will be screened for replication defects in human vascular endothelial cells and phenotypic variants will be characterized for defects in a mouse model of Spotted Fever pathogenesis.